Combination antiretroviral therapy (ART) results in marked suppression of viremia in persons with HIV-1 infection. Therapy is not curative, however, and detectable viremia and replication-competent HIV-1 persist despite ART-induced suppression. The origin of persistent viremia on therapy is uncertain; potential sources include ongoing complete cycles of HIV-1 replication, long-lived reservoirs of chronically infected cells, sanctuary sites into which antiretrovirals have poor penetration, or a combination of these possibilities. Understanding the source and mechanisms of viral persistence on ART has critical implications for future therapeutic approaches and strategies for virus eradication. We began our investigation of the source of persistent HIV by developing an assay for viremia (HIV RNA) with single-copy sensitivity and by developing clinical protocols to determine the effects of ART intensification. These studies and others revealed no decrease in persistent viremia after drug intensification, suggesting that persistent viremia may be the product of long-lived reservoirs of chronically infected cells. Others have reported the utility of 2-LTR circles as an indicator of continued HIV replication during ART and increases in such circular forms during intensification with raltegravir. In addition, a survey of anatomic reservoirs revealed a higher HIV RNA to DNA ratio in cells from gut-associated lymphoid tissue (GALT) in the terminal ileum compared to the colon and rectum and decreases in HIV RNA during drug intensification. Such detailed analyses demonstrate the complexities of host-virus interactions and highlight the limitations in our understanding of mechanisms of persistence during suppressive ART. The proposed project represents a focused approach to overcoming our limitations and understanding persistence by quantifying the host and viral contributions to HIV persistence. We are building on prior studies that used sensitive methodologies to quantify and genetically characterize virus in plasma to investigate HIV reservoirs in cellular compartments, and are now expanding the range of our analyses by applying new single-cell methodologies and isolating specific cell subsets in blood and tissue from infected individuals. To characterize the host-virus relationship during suppressive therapy, we are quantifying cellular and soluble immune correlates of persistent viremia. We initiated these studies by investigating the level of cellular immune activation before and after initiation of ART. The relative proportion of cellular immune activation markers (e.g., CD8+CD38+DR+cells) were high prior to therapy, but declined sharply after ART was initiated; ultimately, HIV RNA levels and levels of immune activation stabilized to a persistent steady state with approximately the same time frame. Previous investigators detected persistent cellular immune activation during ART, but analyses to date have been restricted to 2-3 years on ART. To determine whether immune activation was still elevated after achieving steady-state persistent viremia, we quantified levels of cellular immune activation markers in patients with viremia suppressed for more than7 years on ART and age-, sex-, and race-matched uninfected controls. A modest but significant level of cellular immune activation (CD8+CD38+DR+ cells) was detectable even after 7 years on ART. We investigated potential causes of persistent immune activation first by characterizing PBMC more fully, quantitating memory and naive, CD38, and DR subsets in CD4 and CD8 lineages. In parallel, we quantitated the levels of persistent viremia. Initial evaluation revealed a strong association between levels of persistent HIV RNA and CD8 memory subsets (r=0.51, p=0.0004) and CD8+CD38+DR+ immune activation (r=0.44, p=0.003). These data indicate that either generalized cellular activation itself drives production of HIV or activation is present in response to persistent viremia. Determining the difference between these two possibilities will offer new insights for therapeutic strategies to eliminate such cellular reservoirs. If generalized activation is the source of persistent viremia, then treatment with agents that stimulate the immune system and increase cellular activation will result in increased HIV production from latent reservoirs, followed by overall decay in viremia. In contrast, if increased immune activation is directly controlling the level of persistent viremia, then further immune activation could result in its decay. With the HVIB Translational Research Unit, we will take a dual approach to define further characteristics of CD4+CD38+ and CD8+CD38+ cell subsets by quantifying additional markers in the long-term suppressed patients. Our prediction is that CD8 markers of activation and proliferation will correlate with viremia, but CD4 markers will not. If so, we will have identified a key distinction between cellular immune activation and viremia before and after introduction of ART. We are further characterizing the CD4 cell population by quantitating subsets of CD25+FoxP3+ (suppressor T reg) and IL-17+ (helper cells). Our hypothesis is that persistent viremia will be positively correlated with the relative frequency of FoxP3+ cells because these cells have immunosuppressive function, resulting in higher levels of HIV-1. We are also using our well-characterized group of patients with long-term suppressed viremia on ART to characterize soluble markers of inflammation as correlates of viremia. Levels of soluble markers, such as D-Dimer, IL-6, C-reactive protein all cause mortality in HIV infection, even after suppression on ART. There are no data correlating the relative levels of HIV viremia to predictive outcome markers. We will determine whether levels of soluble markers correlate with persistent viremia. We will also quantify the effects of immune responses on HIV genetic variation. Using single-genome sequencing (SGS) techniques, we will determine whether prolonged viral suppression and partial immune reconstitution result in selection for cells infected with HIV immune escape variants. These studies will provide the first fine-structure analysis of HIV populations during prolonged ART. In addition, we have initiated several new studies of HIV persistence. We are investigating HIV in plasma, PBMC, and cells derived from ileum and colon in infected individuals taking combination ART with suppressed 50 copies who are undergoing colonoscopy at the NIH Clinical Center. We have performed these colonoscopies in collaboration with J. Kovacs in the protocol Virologic and Immunologic Evaluation of Lymph Node, Tonsillar and Intestinal Biopsies, and Bronchoalveolar Lavage Fluid and have used a new sampling strategy that will yield useful information regarding the distribution of HIV-infected cells in the gastrointestinal tract. In a second study of HIV persistence, we are studying HIV from plasma and PBMC from patients with viral RNA suppressed on ART who undergo short antiretroviral discontinuation. Using SGS to investigate HIV from the earliest rebound viremia occurring within 7-14 days of discontinuation, we will identify a critical source of viremia. In collaboration with S. Hughes (DRP) , X. Wu (Leidos), J. Coffin (Tufts), M. Kearney (DRP), and J. Mellors (University of Pittsburgh), we have investigated HIV integration sites in vivo, characterizing HIV from plasma and PBMC of patients. Drs. Hughes and Wu have completed analysis of integration sites in these individuals. These studies revealed that specific integration sites may be linked to clonal expansion of HIV-infected cells, suggesting a novel mechanism for HIV persistence. [Corresponds to Project 1 in the October 2011 site visit report of the Clinical Retrovirology Section, HIV Drug Resistance Program]